The cytokines of the interleukin-6 family are multifunctional proteins that regulate cell growth, differentiation, and other cell functions in a variety of biological systems including the immune, inflammatory, hematopoietic, and nervous systems. One member of this family, ciliary neurotrophic factor (CNTF), displays biological functions more restricted to the neuromuscular axis. We have recently identified two additional ligands for the CNTF receptor complex. Both are composite cytokines formed by cardiotrophin-like cytokine (CLC) associated to either the soluble type I cytokine receptor CLF or the soluble form of CNTF receptor alpha (CNTFRalpha). The present study was aimed at analyzing the interactions between the cytokine CLC and its different receptor chains. For this purpose, we modeled CLC/receptor interactions to define the residues potentially involved in the contact sites. We then performed site-directed mutagenesis on these residues and analyzed the biological interactions between mutants and receptor chains. Importantly, we found that CLC interacts with the soluble forms of CNTFRalpha and CLF via sites 1 and 3, respectively. For site 1, the most crucial residues involved in the interaction are Trp67, Arg170, and Asp174, which interact with CNTFRalpha. Surprisingly, the residues that are important for the interaction of CLC with CLF are part of the conserved FXXK motif of site 3 known to be the interaction site of LIFRbeta. Obtained results show that the Phe151 and Lys154 residues are effectively involved in the interaction of CLC with LIFRbeta. This study establishes the molecular details of the interaction of CLC with CLF, CNTFRalpha, and LIFRbeta and helps to define the precise role of each protein in this functional receptor complex.